EFFECTS OF CYTOCHALASIN, PHALLOIDIN, AND PH ON THE ELONGATION OF ACTIN-FILAMENTS
EFFECTS OF CYTOCHALASIN, PHALLOIDIN, AND PH ON THE ELONGATION OF ACTIN-FILAMENTS
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DOI：
10.1021/bi00221a034
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发表时间：
1991-02-19
期刊：
影响因子：
2.9
通讯作者：
POLLARD, TD
中科院分区：
文献类型：
--
作者：
SAMPATH, P;POLLARD, TD
我们用电子显微镜测量了细胞松弛素、鬼臼糖苷和pH对肌动蛋白细丝带刺和尖端伸长率的影响。在细胞松弛素的情况下，我们比较了对ATP-和ADP-肌动蛋白单体的影响。微摩尔浓度的细胞松弛素B(CB)或细胞松弛素D(CD)抑制了丝状体两端的伸长，在带刺的一端约为95%，在尖端约为50%，因此两端对生长速度的贡献大致相等。ATP-肌动蛋白和ADP-肌动蛋白分别在0.1mU-MCB和0.02mU-MCd和0.1mU-MCd对带刺末端的伸长有半最大抑制作用。在尖端，Cd对ATP-肌动蛋白和ADP-肌动蛋白的延长抑制作用大致相同。在高浓度(2-mU-M)时，细胞松弛素同时降低ADP-肌动蛋白和ATP-肌动蛋白的结合和解离速率常数，因此它们对临界浓度的影响很小。这些观察结果证实并推广了Bonder和Mooseker[Bonder，E.M.，&Mooseker，M.S.(1986)J.Cell Biol]的观点。102、282-288]。延长率与细胞松弛素和肌动蛋白浓度的依赖关系可以通过包括细胞松弛素与肌动蛋白单体结合的影响的机制来定量解释[GoDette，D.W.，&Frieden，C.(1986)J.Biol]。Chem，261,5974-5980]和由ADP-肌动蛋白和细胞松弛素的复合体组成的细丝的带刺末端的部分帽。Phalloidin将两端的解离速率常数降低到接近于零，并将带刺末端的缔合速率常数降低约50%。这证实并推广了Coluccio和Tilney[Coluccio，L.M.，&Tilney，L.G.(1984)J.Cell Biol]的观察结果。99,529-]，并提供了令人信服的证据，表明鬼臼毒素影响亚单位结合和解离。在pH为6.6-8.3范围内，pH对带刺末端的缔合速率常数影响很小，但在碱性pH时，解离速率常数较大。在尖端，缔合速率常数在碱性条件下略有下降。总而言之，这些影响解释了在碱性PHS下较高的临界浓度和较慢的聚合速度。
We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1-mu-M CB and 0.02-mu-M CD for ATP-actin and at 0.1-mu-M CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2-mu-M) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem, 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin. Phalloidin reduces the dissociation rate constants at both ends to near zero and also reduces the association rate constant at the barbed end by about 50%. This confirms and extends the observations of Coluccio and Tilney [Coluccio, L. M., & Tilney, L. G. (1984) J. Cell Biol. 99, 529-535] and provides convincing evidence that phalloidin affects both subunit binding and dissociation. Over the pH range of 6.6-8.3, the pH has very little effect on the association rate constant at the barbed end, but the dissociation rate constant is larger at alkaline pH. At the pointed end, the association rate constant decreases slightly at alkaline pH. Together, these effects account for the higher critical concentration and slower rates of polymerization at alkaline pHs.